This section introduces embodiments that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
Liposomes are small artificial vesicles of spherical shape having one or more phospholipid bilayers. Due to their size and hydrophobic and hydrophilic character (besides biocompatibility), liposomes are promising systems for drug delivery, nanopore sensing platform technology and other vast applications in modern biotechnologies. Liposome properties differ considerably with lipid composition, surface charge, size, and the method of preparation.
A proteoliposome is a liposome into which one or more proteins have been inserted, wherein the protein maintains its biological functions (M. Scalise, et al., Pharmaceutics 2013, 5, 472-497). Those inserted proteins, including membrane transporter proteins, viral portal proteins, and other hydrophobic transmembrane channel proteins, can be structurally and functionally investigated.
A viral portal protein is a protein embedded in the capsid shell in bacteriophages, which serves as a conduit for single stranded DNA, single stranded RNA or double stranded DNA to pass through. A membrane channel protein, or a protein channel, is a protein that enables the transport of specific substances across a cell membrane. Protein channels are a traffic system in a cell that allows the transportation of water, chemicals and electric signals across the cell membrane. They affect the function of the cell by controlling the traffic of the materials and signals. More specialized protein channels transport calcium, sodium and other ions to change the electrical potential across cell membranes causing the cells to react to stimuli. A proteoliposome with integrated membrane channel proteins is an ideal man-made system to study those biological processes of living cells. Furthermore, a proteoliposome with a single portal protein molecule could fuse quickly and efficiently to a planar lipid bilayer for diverse nanopore sensing applications.
The planar lipid bilayer technology is a technique that yields incredibly useful structural-functional information about a single channel protein. It is also currently actively utilized as a powerful platform using biological protein nanopore for the development of single-molecule nanopore sensing technology, as well as ultrafast DNA sequencing technology. Portal protein GP10 from the bacteriophage ϕ29 was the first phage portal protein shown to be successfully inserted into planar bilayer membranes, thereby it may inspire more researchers to apply the techniques to portal proteins from the other bacteriophages (D. Wendell, et al., Nat Nanotechnol, 2009, 4, 765-772). More recently, the technique has been further explored as a single-molecule sensing platform for the development of nanopore sensors (W. Li, et al., Angew Chem Int Ed Engl, 2013, 52, 4350-4355; S. Majd, et al., Current Opinion in Biotechnology, 2010, 21, 439-476) and ultrafast DNA sequencing technology (H. Bayley, Phys Life Rev, 2012, 9, 161-163; discussion 174-166; M. Wanunu, Phys Life Rev, 2012, 9, 125-158).
However, insertion of a channel protein into planar bilayer membrane is technically difficult as well as time-consuming. Before the fusion of phage portal protein into the planar bilayer membrane, the portal protein is first reconstituted and incorporated in proteoliposomes. Most of the phage portal proteins have low solubility, and may self-aggregate during the preparation of the proteoliposomes. Furthermore, the fusion of the formed proteoliposomes to the planar bilayer membrane is sporadic, unpredictable and varied significantly from experiment to experiment. Due to the lack of experimental consistency between laboratories, the results from different methodologies reported for generating fusible proteoliposomes are highly variable. To enable wide and practical uses of the planar lipid bilayer and the single-molecule nanopore sensing technologies, there are unmet needs for a simple, practical, and economically viable preparation of proteoliposomes, which could be effectively and efficiently fused into a planar lipid bilayer.